Natural products continue to serve as a key platform for drug development, many of which are decorated with essential sugar residues. Weymouth-Wilson AC (1997) Nat Prod Rep 14: 99-110. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels and/or even the molecular mechanism of action. Thorson J S, et al. (2002) in Carbohydrate-Based Drug Discovery, ed. Wong C-H (Wiley-VCH, Weinheim), pp. 685-712; Ahmed A, et al. (2006) J Am Chem Soc 128: 14224-5. As an emerging method to differentially glycosylate natural products, glycorandomization employs the inherent or engineered substrate promiscuity of anomeric kinases (FIG. 1A, E1), and nucleotidyltransferases (E2), for the in vitro synthesis of sugar nucleotide libraries as potential sugar donors for suitable natural product glycosyltransferases (GTs). Hoffmeister D, et al. (2003) Proc Natl Acad Sci USA 100: 13184-9; Yang J, et al. (2005) Chem Biol 12: 657-64; Barton W A, et al. (2002) Proc Natl Acad Sci USA 99:13397-402; Griffith B R, et al. (2005) Curr Opin Biotechnol 16: 622-30. The successful glycorandomization of various natural product scaffolds has been reported including the antibiotic vancomycin, the antihelmenthic avermectin, and the anticancer agent calicheamicin. Fu X, et al. (2003) Nat Biotechnol 21: 1467-9; Zhang C, et al. (2006) J Am Chem Soc 128: 16420-1; Zhang C, et al. (2006) Science 313: 1291-4. In contrast, other recent antibiotic glycorandomization studies revealed novobiocin and erythromycin GTs, (NovM and EryBV), to accept only 2 alternative sugar nucleotides out of 25-40 potential donors tested. Albermann C, et al. (2003) Org Lett 5: 933-6; Zhang C, et al. (2007) Chembiochem 8: 385-390. Thus, while permissive GTs open new opportunities for drug discovery, the stringent specificity of other GTs remains a limiting factor in natural product diversification and highlights a need for general GT engineering and/or evolution platforms.
GTs constitute a large family with currently ˜23,000 predicted or known GT sequences in the CAZY database divided into 87 families based upon amino acid similarity. Despite the vast range of GT sugar donors and acceptors (sugars, proteins, nucleic acids, lipids, and small molecules), GTs are generally classified into two simple groups based upon mechanism (inverting or retaining), and primarily fall within two main structural superfamilies (GT-A and GT-B). Lairson L L, et al. (2004) Chem Commun 2243-8; Hu Y., et al. (2002) Chem Biol 9: 1287-96. The GT-B fold is the predominate fold of natural product GTs and is characterized by two closely associated Rossman-like domains, each of which is usually distinguished as the acceptor- and donor-binding domains (N and C-terminal domains, respectively). Despite the wealth of GT structural and biochemical information, attempts to alter GT donor/acceptor specificities via rational engineering have been largely unsuccessful and primarily limited to sequence-guided single site mutagenesis. Hancock S M, et al. (2006) Curr Opin Chem Biol 10: 509-19. While there exists precedent for the directed evolution of carbohydrate-utilizing enzymes, the lack of sensitive high-throughput screens for GTs has also hampered GT directed evolution. Hoffmeister D, et al. (2003) Proc Natl Acad Sci USA 100: 13184-9; Williams G J, et al. (2006) J Am Chem Soc 128: 16238-47. Withers et al recently described a unique in vivo selection for the directed evolution of the bifunctional sialyltransferase CstII, the structure of which closely resembles those of the GT-A superfamily, and has recently been suggested to be classified into a third structural superfamily (GT-C). Aharoni A, et al. (2006) Nat Methods 3: 609-14; Chiu C P, et al. (2004) Nat Struct Mol Biol 11: 163-70; Breton C, et al. (2006) Glycobiology 16: 29R-37R. The CstII directed evolution study relied upon trapping a fluorescently-tagged acceptor sugar inside E. coli upon modification by the negatively-charged sialic acid as an in vivo screen. Yet, there remains a lack of successful high throughput screens or directed evolution studies targeted toward the structurally distinct and functionally important GT-B family.
As can be appreciated, glycosyltransferases possessing expanded substrate specificities are desirable and would greatly benefit the production of, for example, diverse chemical libraries containing glycosylated compounds possessing novel and/or improved bioactivities.